Field of the Invention
The invention generally relates to compositions and methods to increase the ability of hypoxia-activated bioreductive agents to kill tumor cells within solid tumors. In particular, the invention provides methods and compositions to create local regions of hypoxia within a tumor, or within a region containing a tumor, in order to enhance activation of and tumor cell killing by hypoxia-activated bioreductive agents within the local region.
Background of the Invention
Tumor growth requires the development of a network of neovasculature to supply oxygen and nutrients and to remove toxic metabolites. The neovasculature in tumors differ significantly from normal vasculature (1, 2). Tumor neovasculature is abnormal, chaotic and inadequate in structure and function, and some data suggest that tumor vasculature may rely more on tubulin as the cytoskeletal support in contrast to both tubulin and actin as the cytoskeleton in normal tissue (3). Targeting tumor vasculature has evolved into a useful strategy to develop new cancer therapeutics (4). Two approaches are currently used to target tumor vessels. One is to prevent the angiogenic process by blocking angiogenic factors or their receptors in order to prevent new vessel formation. This type of therapy is represented by bevacizumab, a monoclonal antibody against vascular endothelial growth factor (VEGF), and sorafenib or sunitinib, small molecular inhibitors of VEGF receptor tyrosine kinase (4-8).
The second strategy of targeting tumor vasculature is to kill the existing endothelial cells in the tumor directly. This group of compounds is referred to as vascular disrupting agents (VDAs) (9, 10). Their goal is to kill the endothelium of existing tumor vessels to deprive tumors from getting an adequate blood supply, leading to tumor ischemia and eventually tumor necrosis. This group of agents is represented by several small molecules that include combretastatin A4 (CA4), ZD6126, AVE8062, Oxi4503 and stilbene derivatives (9-13). These small molecules kill tumor endothelial cells by interfering with microtubule polymerization at the colchicine site. Several colchicine-site microtubulin inhibitors are currently in development as VDAs.
Induction of Tumor Hypoxia and Development of Compensatory Responses
With both anti-angiogenic agents and vascular disrupting agents, the central theme of these tumor vessel targeting agents is to deprive tumor cells of vascular support so that the tumor develops hypoxia and then undergoes necrosis. Development of hypoxia in a tumor is thus the key requirement to induce tumor cell death. However, hypoxia in tumors is not sufficient to induce cell death because hypoxic tumor cells develop a variety of hypoxia responses such as stabilization of Hypoxia-Induced Factor (HIF) 1-α (14, 15), which induces production of glycolysis enzymes that promote survival in the hypoxic environment, or generation of VEGF and other angiogenic factors to induce angiogenesis. Nitric oxide (NO), another factor that is produced in tumor cells during hypoxia, induces vasodilatation and thus also improves tumor blood supply (16, 17). NO is also tightly linked to angiogenesis (18, 19). These compensatory mechanisms can thus result in drug resistance against anti-angiogenic agents and VDAs.
Strategies to Enhance Therapeutic Efficacy
There are a few potential strategies to enhance therapeutic efficacy of targeting tumor vasculature. One is to combine either group of agents with conventional chemotherapy, which is currently a common strategy used with anti-angiogenic agents. Bevacizumab, a monoclonal antibody against VEGF, is typically combined with chemotherapy for treating colorectal cancer, non-small cell lung cancer and breast cancer. VDAs are currently in phase II trials for various solid tumors but none have received FDA approval thus far. One new strategy is to combine anti-angiogenic agents with VDAs. This is based on the observation that VDAs induce elevation of VEGF, which subsequently mobilizes bone marrow endothelial progenitor cells into circulation and is thus responsible for repair of damaged tumor vessels (20). Using an inhibitor of the VEGF pathway could possibly block the mobilization and enhance therapeutic effects (11, 20), but this strategy has not been proved in clinical setting.
Tirapazamine (SR4233; 3-amino-1,2,4-benzotriazine-1,4-di-N-oxide) is a bioreductive agent that works exclusively in hypoxic environments and has also been tested as an anticancer agent (21). Tirapazamine is activated by cytochrome P450 reductase by a one-electron reaction, thereby generating nitroxide radicals. In the absence of oxygen, nitroxide radicals induce single- and double-strand breaks in DNA to cause cell death. Because of this property, tirapazamine exhibits 15-200-fold greater toxicity to hypoxic cells compared to well-oxygenated cells. This agent has also been shown to be a radiation sensitizer and to act synergistically with platinum compounds in cancer therapy regimens (22, 23).
Mechanism of Action for Tirapazamine
A postulated mechanism of action of tirapazamine is shown in the following figure (24-28). One electron reduction of tirapazamine proceeds through reductive activation by enzymes including cytochrome P-450, NADH-cytochrome P-450 reductase and other flavo- or metalloproteins. The product for the single electron transfer reaction is a free radical (I or II), which can be oxidized by oxygen to yield a superoxide and the parent drug tirapazamine. Alternatively, the free radical (I or II) can acquire a second electron through a hydrogen abstraction reaction intermediate (III) to form a stable mono-N-oxide (SR4317). These divergent fates lead to the selective metabolism of tirapazamine in the hypoxic environment. The acquisition of the second electron from macromolecules was suggested to be the cause of lethal damage to hypoxic cells. Another possible route is the release of a hydroxyl radical from intermediate II to produce SR4317 directly, which can be further metabolized in a similar reaction to SR4330.

Among all organs, liver is the most important organ for the metabolism of tirapazamine. Costa et al. examined the toxicity of tirapazamine in cultured rat hepatocytes that were transiently incubated in a hypoxic environment at oxygen concentrations of 1, 2, 4, 10 and 20% (29). The dose response curve of tirapazamine to monolayer hepatocytes shifted significantly to the left, meaning more susceptible to death, as the oxygen concentration decreased (P<0.05). The concentration of tirapazamine that caused 50% cell death over 2 h at 4% oxygen was more than 10-fold less than that required at 10% or 20% oxygen concentration. The concentration of tirapazamine required to induce 50% cell death at 2% oxygen was 15-fold less than that required to induce same degree of cell death at 20% oxygen. When the oxygen concentration was further decreased to 1%, the concentration of tirapazamine required for 50% cell death is 50-fold lower than that required at 20% oxygen. These results indicate that the potency of tirapazamine was 15 and 50-fold stronger in 2% and 1% oxygen, respectively, compared with that in 20% normal oxygen environment.
Preclinical and Clinical Development of Tirapazamine
Significant development of tirapazamine has been done both pre-clinically and clinically. Animal studies of tirapazamine showed that its potential side effects include bone marrow toxicity, necrosis of the olfactory nerve, and retinal degeneration (21). A phase I clinical study of tirapazamine by intravenous administration every three weeks showed a maximum tolerated dose (MTD) of 390 mg/m2, and dose limiting toxicities include reversible ototoxicity and transient visual abnormality when the dose was above 330 mg/m2 (30, 31). Other non-specific toxicities included muscle cramps, nausea, vomiting, and diarrhea. Grade 1 thrombocytopenia was noted in one patient receiving 450 mg/m2 and no leukopenia was noted in any patient (30).
Phase II studies of tirapazamine have been carried out for lung cancer, cervical cancer, ovarian cancer, melanoma and head and neck cancer with promising results (32-35). In the phase III randomized study of stage IV non-small cell lung cancer, 367 patients were randomized to receive carboplatin and paclitaxel with tirapazamine (260 mg/m2 in cycle 1 and increased to 330 mg/m2 in cycles 2-6 if tolerated in cycle 1) (n=181); or carboplatin and paclitaxel with a placebo (n=186). Unfortunately, the result was disappointing in that there was no benefit in response rate, overall survival, or progression free survival in the group that received tirapazamine. In contrast, increased systemic toxicity such as abdominal cramps, fatigue, transient hearing loss, febrile neutropenia, hypotension, myalgias, and skin rash were observed and led to significant drop out from the study (36). The trial was terminated early after interim analysis, which showed that it would not reach the predicted 37.5% improvement in survival. Another large phase HI randomized study in pelvis-confined cervical cancer is ongoing for patients receiving cisplatin and radiation with or without tirapazamine, and the result is currently unavailable.
Clearly, there is an ongoing need to provide improved cancer treatments. In particular, it would be beneficial to provide cancer therapy protocols using known agents in a manner that optimizes their efficacy and minimizes deleterious side effects.